DNA mitocondrial é alvo de integração do HBV

Biologia das Comunicações, volume 6, número do artigo: 684 (2023) Citar este artigo

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O vírus da hepatite B (HBV) pode integrar-se ao genoma das células infectadas e contribuir para a hepatocarcinogênese. No entanto, o papel da integração do VHB no desenvolvimento do carcinoma hepatocelular (CHC) permanece obscuro. Neste estudo, aplicamos uma abordagem de sequenciamento de integração de HBV de alto rendimento que permite a identificação sensível de locais de integração de HBV e a enumeração de clones de integração. Identificamos 3339 locais de integração do HBV em amostras de tecidos tumorais e não tumorais pareados de 7 pacientes com CHC. Detectamos 2.107 integrações clonais expandidas (1.817 em tumores e 290 em tecidos não tumorais), e um enriquecimento significativo de integrações clonais de HBV no DNA mitocondrial (mtDNA) ocorrendo preferencialmente nos genes de fosforilação oxidativa (OXPHOS) e na região D-loop. Descobrimos também que as sequências de RNA do HBV são importadas para as mitocôndrias das células do hepatoma com o envolvimento da polinucleotídeo fosforilase (PNPASE), e que o RNA do HBV pode ter um papel no processo de integração do HBV no mtDNA. Nossos resultados sugerem um mecanismo potencial pelo qual a integração do HBV pode contribuir para o desenvolvimento do CHC.

A infecção crônica pelo vírus da hepatite B (HBV) é um importante fator de risco para o desenvolvimento de carcinoma hepatocelular (CHC). Em comparação com indivíduos saudáveis, os pacientes com hepatite B crônica (CHB) apresentam risco até 100 vezes maior de desenvolver CHC, que é a quarta principal causa de morte relacionada ao câncer em todo o mundo, com ~780.000 mortes/ano1,2. O ADN viral integrado foi detectado em 85-90% dos CHC relacionados com o VHB, e a sua presença em tumores que se desenvolvem nos fígados não cirróticos de crianças ou adultos jovens apoia ainda mais o papel da integração do ADN viral na hepatocarcinogénese3,4,5, 6,7. A integração do DNA do HBV no genoma do hospedeiro pode levar à instabilidade cromossômica, mutagênese insercional, desregulação da expressão do gene hospedeiro e produção de proteínas virais mutantes, como proteínas de superfície truncadas e HBx com propriedades oncogênicas conhecidas . Embora os locais de inserção do DNA do HBV pareçam estar distribuídos aleatoriamente por todo o genoma do hospedeiro, o uso recente de abordagens de sequenciamento de próxima geração (NGS) levou à identificação do enriquecimento da integração do HBV em genes específicos do câncer, incluindo TERT, MLL4, CCNE1 e CCNA2, em tecidos tumorais7,10,11,12,13,14,15,16. Além disso, um estudo recente mostrou que alterações recorrentes no número de cópias em genes causadores de câncer podem estar associadas à integração viral distante16. Embora um número significativo de casos tenha sido estudado, genes conhecidos relacionados ao câncer são alterados pela integração do VHB em apenas uma pequena proporção de CHCs relacionados ao VHB. Numerosos estudos também demonstraram que elementos genômicos específicos, como sequências repetitivas, sequências de DNA para RNAs não codificantes e retrotransposons, são alvo da integração do HBV7,11,12,14,17,18,19. Um estudo em Hong Kong que analisou linhas celulares de CHC positivas para HBV mostrou que a expressão de um transcrito quimérico específico de HBx-LINE1 tem uma função promotora de tumor, com uma grande proporção dos CHC avaliados expressando este transcrito . No entanto, a expressão do HBx-LINE1 não foi confirmada em uma grande série de CHCs relacionados ao HBV de pacientes europeus20.

Apesar dos progressos realizados na investigação sobre a integração do ADN do VHB, muitos aspectos importantes permanecem obscuros. No geral, o desenvolvimento de métodos alternativos de detecção para a integração do HBV pode ajudar a obter uma melhor compreensão dos mecanismos envolvidos no processo carcinogénico induzido pela integração do HBV.

Ao aplicar um método de sequenciamento de integração de HBV de alto rendimento (HBIS), neste estudo detectamos enriquecimento da integração do vírus no DNA mitocondrial (mtDNA) de tecidos hepáticos tumorais e não tumorais de pacientes com CHC. Além disso, aplicamos HBIS e RNASeq a mitocôndrias purificadas a partir de células HepAD38 induzidas por HBV e detectamos múltiplas integrações de HBV em mtDNA, bem como transcritos de fusão HBV-mitocondrial. Todas as integrações mitocondriais nos tecidos tumorais foram expandidas clonalmente e envolveram tanto os genes mitocondriais da fosforilação oxidativa (OXPHOS) quanto a região D-loop. Descobrimos também que sequências de RNA do HBV são importadas para as mitocôndrias das células do hepatoma e que a polinucleotídeo fosforilase (PNPASE) pode estar envolvida na importação de transcritos virais.

15 kb, with more than 20% having a length shorter than 100 bp31,46,55. Considering that mtDNA averages several thousand copies per hepatocyte compared to the two copies of numts in nuclear DNA (nDNA), by isolating mitochondria from cells, it is possible to completely dilute out numts, leading to numt-free mtDNA sequences. Therefore, to study mtDNA HBV integration, we isolated nuclei, cytoplasm, and mitochondria from HBV-producing HepAD38 cells and applied both HBIS and RNASeq. According to HBIS, several HBV integration sites were identified in DNA isolated from mitochondria, whereas no integration was detected in numts from nDNA. Furthermore, RNASeq revealed the presence of chimeric HBV-mitochondrial transcripts within mitochondria but not in cytoplasm or nuclei of HBV-producing HepAD38 cells, and mtDNA insertion sites may be transcriptionally active in these cells. Both HBIS and RNAseq analysis also revealed that MMEJ have a major role in HBV integrations occurring in mitochondrial genomes. Therefore, taken together, our data clearly demonstrate that HBV can integrate into mtDNA of tumour and non-tumour hepatocytes. Some previous studies have reported data concerning HBV integration in mtDNA16,56,57,58,59,60. All these studies have utilised high-throughput HBV genome-enrichment sequencing approaches to study HBV integration, and most of them have analysed hepatoma cell lines stably expressing HBV DNA56,57,58,59.To the best of our knowledge, only one16 of the papers has reported data on HBV integration in mtDNA from human liver tissues. In particular, in the supplementary dataset of this paper, 58 different HBV integration sites in mtDNA from tumour and/or non-tumour liver tissue specimens of 11 patients with HBV-related HCC have been listed16. The mitochondrial genomic regions most frequently targeted by HBV integration in the 11 patients were the D-loop region, ND4, ND5, RNR2, CYTB, ND6, ND1, ND2 and COX3 genes16. HBV integration events have also been described in mtDNA from humanised-liver tissue samples of chimeric mice60. In this study, Furuta et al.60 have identified 50 distinct HBV integration sites in mtDNA from chimeric mice. These integrations (a) have been associated with higher levels of HBV replication, (b) occurred at higher frequency in the D-loop region, and (c) appeared to rely on MMEJ60. No detailed information on virus-mtDNA junctions has been provided in studies performed on PLC/PRF/5 cell lines56,59. However, the fact that HBV integration in mtDNA may occur in these cells—which do not replicate HBV and only express multiple distinct viral RNAs from HBV integrants56,59—suggests that viral RNA might be involved in the process of HBV integration in mtDNA. Despite a number of studies documenting interaction between HBV proteins and mitochondria and consequent alteration of mitochondrial functions61,62,63, whether HBV nucleic acids may translocate into mitochondria has only minimally been addressed. Based on our results, HBV transcripts, but not viral full-length genome or cccDNA, can localise to mitochondria. In addition, PNPASE, a mitochondrial protein considered the first RNA import factor for mammalian mitochondria35,36,39, possibly mediates viral RNA delivery into the mitochondrial matrix. A PNPASE-dependent RNA import sequence that we identified for the preS1 transcript as well as known stem-loop structures specific to HBV transcripts appear to mediate mitochondrial targeting of viral RNAs. Localisation of HBV transcripts to mitochondria leads us to hypothesise that viral RNA may represent a possible substrate for HBV integration in mtDNA. The mitochondrial genome is more prone to damage and double-strand break (DSB) formation than the nuclear genome due to frequent exposure to the ROS generated by mitochondrial oxidative phosphorylation and the lack of protective histones. Considering that several reports have shown that RNA molecules can directly act as a template for the repair of mitochondrial DSBs in human cells64, it is tempting to speculate that viral exploitation of this pathway may lead to HBV sequences being inserted into the mitochondrial genome. In summary, we found that HBV may integrate into mtDNA, with tumours and non-tumour liver tissues showing distinct profiles of viral integration into the mitochondrial genome. Moreover, our results indicate that HBV RNA may be actively imported into mitochondria and that viral RNA sequences might be involved in the process of HBV integration into mtDNA. In spite of the relatively limited sample of patients, this study offers new insight into the HBV-hepatocyte interaction and provides a new basis for investigative analyses that may lead to further comprehension of the mechanisms by which HBV insertion can drive HCC development and progression./p> 5 exo− (5000 U/mL) (New England Biolabs, Ipswich, MA) for 1 h at 37 °C. All reactions were purified by a MinElute Reaction Clean-up kit (Qiagen). Each aliquot of blunted, A-tailed DNA fragments was then ligated to 200 pmol annealed linkers (LinkerTop + LinkerBottom) (Supplementary Table 2) with 4 μL pLinker, 5 μL NEB T4 DNA ligase buffer and 1 μL T4 DNA ligase (2 × 106 U/mL, high concentration) (New England Biolabs) for 1 h at 25 °C and then overnight at 16 °C. The ligase was inactivated by incubation at 70 °C for 20 min, and the reactions were purified using a MinElute Reaction Clean-up kit (Qiagen). Finally, all six reactions were pooled, and the pooled linker-ligated DNA was aliquoted into two equal parts to perform semi-nested ligation-mediated PCR with forward or reverse HBV primers (Fig. 1 and Supplementary Table 2). The forward and reverse enrichment sequences were kept separate throughout the remainder of the protocol. The DNA was divided into 1-µg aliquots; each aliquot was mixed with 20 µL Phusion HF buffer (5×), 3 μL dNTPs (10 mM), 1 μL biotinylated forward (20 μM) or reverse HBV primer (Supplementary Table 2) (2.5 μM), 1 μl Phusion Taq (2000 U/ml) (New England Biolabs) and H2O to 50 μL. Single-primer PCRs were performed as follows: 98 °C for 1 min; 12 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 45 s; 72 °C for 1 min); and a hold at 4 °C. Each tube was then spiked with 1 μL pLinker (Supplementary Table 2) (2.5 μM) and subjected to additional cycles of PCR, as follows: 98 °C for 1 min; 35 cycles of 98 °C for 15 s, 65 °C for 30 s and 72 °C for 45 s; 72 °C for 5 min; and a hold at 4 °C. Forward and reverse PCRs were purified using the QIAquick PCR purification kit (Qiagen). The purified products were well separated on a 2% agarose gel, and fragments of 300–1000 bp were excised. The DNA was purified using a QIAquick gel extraction kit, and gel-based size selection and purification was repeated once. Then, 100 μL T1 magnetic streptavidin beads (Invitrogen) were added to each forward and reverse PCR product, and the mixture was incubated for 1 h with gentle rocking at room temperature. The beads were magnetically isolated, washed three times in 500 μL 1× B&W buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl) and once in H2O and resuspended in 50 μL H2O. Subsequently, 25 μL of the beads from each of the forward and reverse PCRs were separately mixed with 10 µL Phusion HF buffer (5×), 1.5 μL dNTPs (10 mM), 1 μL forward (20 µM) or reverse MiSeq HBV primer (20 μM), 1 μL forward or reverse MiSeq-pLinker (20 μM) (all MiSeq primers contain an adaptor for Illumina flow cell surface annealing) (Supplementary Table 2), 0.5 μL Phusion Taq (2000 U/mL), and 11 μL H2O and subjected to PCR (98 °C for 1 min, 35 cycles of 98 °C for 10 s, 65 °C for 40 s, and 72 °C–40 s, followed by 72 °C for 5 min and a hold at 4 °C). The PCR products were magnetically separated from the beads and purified using the QIAquick PCR purification kit (Qiagen). The adaptor-ligated fragments were enriched by 25 cycles of PCR with Illumina primers Index 1 and Index 2, as follows: 98 C° for 1 min, 25 cycles of 98 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s), and 72 C° for 5 min. Forward and reverse libraries for the same sample were mixed in equimolar ratios and sequenced by 250-bp paired-end sequencing using an Illumina MiSeq. A total of 340 integration libraries were constructed from liver tissue samples of the nine individuals analysed (7 patients with HBV-related HCC and 2 HBsAg-negative subjects as a control) and from the PLC/PRF/5, HepAD38 and Vero cell lines./p>

3.0.CO;2-E" data-track-action="article reference" href="https://doi.org/10.1002%2F1097-0142%28195405%297%3A3%3C462%3A%3AAID-CNCR2820070308%3E3.0.CO%3B2-E" aria-label="Article reference 24" data-doi="10.1002/1097-0142(195405)7:33.0.CO;2-E"Article CAS PubMed Google Scholar /p>

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